Download Thyroid Gland Explant Culture

A Systems Approach to
Characterizing and Predicting
Thyroid Toxicity
Michael Hornung, Kara Thoemke,
Joseph Korte, Jose Serrano, John Nichols,
Patricia Schmieder, Joseph Tietge, Sigmund Degitz
US EPA, Mid-Continent Ecology Division, Duluth, MN
McKim Conference
June 27-29, 2006
Duluth, MN
Thyroid Toxicity Research
 Endocrine Disruptors
 Thyroid hormone is important for growth and development,
neurodevelopment, metabolism
 To understand thyroid toxicity need to look at it in the context of
the whole Hypothalamus-Pituitary-Thyroid Axis (HPT)
Thyroid Hormone Regulation
Hypothalamus
(CRH)
TRH
(-)
Pituitary
Thyrotropes
T4
TSH
Thyroid Gland
Thyroglobulin
TPO
DIT MIT
colloid
NIS
Iodine
DIT
DIT
Follicular cells
T4
T4
Transthyretin
Deiodination (D2)
Inactivation/
Elimination
Deiodination (D3)
Conjugation
T4
Deiodination
(D2)
Liver
T3
T3
+
TR/RXR
DNA
mRNA
Peripheral Tissue
Thyroid-axis Systems Model
QSAR and
in vitro Models
Organismal
Outcomes
Systems
Model
Hypothalamus
TRH
(CRH)
Pituitary
Retarded
Development
Thyroid Gland
Hypertrophy
(-)
100
Treated
TSH
Thyroid Follicular Cell
Thyroid Gland
50
0
Thyroglobulin
TPO
100
Control
MIT
DIT
50
0
Iodine
T4
DIT
Development
Transthyretin
Inactive TH
Deiodination
Deiodination
Inactive
TH Conjugation
Deiodination
Liver
T3+TR/RXR
DNA
mRNA
Peripheral Tissues
Why an amphibian model ?
 Metamorphosis is controlled by thyroid hormone
 Simple apical endpoint to monitor disruption in vivo
 Molecular events are well characterized
 Easy to raise and test in the laboratory
• Xenopus laevis
Xenopus Metamorphosis
X. laevis Plasma Thyroid Hormones
Leluop and Buscaglia 1977
8
6
Plasma TH(ng/ml)
Climax
T4 ng/ml
T3 ng/ml
7
Prometamorphosis
5
4
3
2
1
0
52
54
56
58
60
NF Stage
62
64
66
68
MED Thyroid Project Objectives
 Conduct studies with known HPT disruptors
 Inhibitors of thyroid hormone synthesis
• Thyroid Peroxidase: Methimazole, Propylthiouracil
• Sodium Iodide Symporter: Perchlorate
 Develop diagnostic measures
 What are the appropriate tissue level endpoints?
• Histology, T4, TSH
 Can gene and protein expression be used as indicators of
thyroid axis disruption?
 Develop assays to enable ranking and prioritization of chemicals
Effect of Methimazole on Development and
Thyroid Histology
14 d Exposure
100
50 mg/L
*
50
25 mg/L
12.5 mg/L
Proportion in stage
0
100
*
50
0
100
50
0
100
Control
50
0
55 56 57 58 59 60
Developmental
Stage
day 8
Summary of Metamorphosis Assay
 X. laevis is sensitive to model thyroid pathway modulators
 Methimazole, 6-PTU, Perchlorate
 Early stage tadpoles (stg 51-54) can be arrested in development
by T4 synthesis inhibitors, stage 60 is not
 Thyroid histology is an essential component of assay
 More sensitive than developmental rate (d8)
 Diagnostic
Diagnostic Research Approach
 Link Chemical-Biomolecular Interaction to Organism Response
 Examine gene expression during normal metamorphosis and
following chemical exposure
 Examine protein changes
 Circulating T4 and TSH
 Responses of tissues isolated from compensatory mechanisms
 Pituitary explant culture: TSH – T4 feedback
 Thyroid explant culture: TSH stimulation, chemical inhibition
of T4 release
 Develop computational – predictive approaches
In vivo Pituitary Gene Expression:
Thyroid Stimulating Hormone
Developmental Expression
Chemical Exposure
8
1.4e+6
6
Copies TSH/ACT
Copies TSH per l RNA Extract
1.6e+6
1.2e+6
1.0e+6
8.0e+5
6.0e+5
Control
Methimazole
Perchlorate
Propylthiouracil
4
2
4.0e+5
2.0e+5
0
0.0
50
51
52
53
54
55
56
57
58
59
60
Developmental Stage
61
62
63
64
65
Day 0.5
Day 1
Day 1.5
Day 2
Day 4
Day 6
In Vivo Thyroid Gland Gene Expression
Sodium/Iodide Symporter
Developmental Expression
Chemical Exposure
0.18
0.10
0.16
Copies NIS/Copies ACT
Copies NIS per Actin
0.08
0.06
0.04
0.02
0.14
Control
Methimazole
Perchlorate
Propylthiouracil
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.00
50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Developmental Stage
Day 0.5
Day 1
Day 1.5
Day 2
Day 4
Day 6
Pituitary Explant Culture
Objective:
Characterize function of the pituitary during development and
the relationship between T4 and TSH
Method:
Culture pituitaries from tadpoles at multiple stages of
development
Measure TSH expression in the pituitaries
Gene expression or T4 release in thyroid glands treated
with media conditioned by pituitary culture
Pituitary Explant Culture
TSH mRNA is repressed by T4
5
TSH/RPL32
4
freshly dissected pituitary
cultured 96 hours without hormone
cultured 96 hours with 1 pM T4
cultured 96 hours with 10 pM T4
cultured 96 hours with 100 pM T4
cultured 96 hours with 1 nM T4
cultured 96 hours with 10 nM T4
3
2
*
1
0
*
**
Stage 54
Stage 58
Stage 62
Stage 66
 Negative feedback mechanism is functional throughout development
although the setpoint changes

sensitivity to T4 decreases
Thyroid Gland Explant Culture
Objective:
Define thyroid-specific outputs in response to TSH and
xenobiotics in the absence of whole organism compensatory
response
Method:
Culture thyroid glands from prometamorphic tadpoles and
treat with TSH and T4 synthesis inhibitors
Measure T4 release and gene expression
Thyroid Gland Explant Culture:
Time relationship of T4 release inhibition
T4 Released (ng T4 / mm3 gland / 24h)
400
1000 ng TSH/ml
1000 ng TSH/ml + MM1
2000 ng TSH/ml
2000 ng TSH/ml + MM1
300
200
100










0
1
2
3
4
5
Day
6
8
10
12

Pituitary Explant Culture
 Feedback mechanisms in the pituitary
• Negative feedback by T4 on the pituitary is present in metamorphosis
• Sensitivity of the pituitary to this inhibition decreases over time
- in early metamorphosis prevent excess T4
- allow more T4 later to complete metamorphosis
Thyroid Explant Culture
Interpretation of compensatory and direct effects
In vitro…
•
Release T4 in response to TSH is dose related
•
T4 reserves must be depleted before synthesis inhibition significantly
affects T4 release
In vivo…
•
Early stages are more sensitive to arrested metamorphosis by T4 inhibitors
than late stages
•
At late prometamorphosis, thyroid glands are larger and reserve T4 is
sufficient to complete metamorphosis
•
Exposure time 0 does not equal effect time 0 for circulating T4
•
Need to measure circulating hormone levels to interpret gene expression
and protein responses in vivo
Potential Endpoints for HPT-Axis QSAR Development
Hypothalamus
TRH/CRH
Tyrosine Iodination
and Hormone Production
Pituitary
TSH
T4 (-)
Thyroid Gland
Iodine Uptake
MIT
I + Tyr
NIS
Iodine
TPO
DIT
DIT
DIT
T4
Metabolizing Enzyme
Induction / Activity
T4
Liver
metabolism/
conjugation
TH-gluc
elimination
T4
Receptor and Protein
Binding
Peripheral Tissue
Deiodination
T3 + TR  T3-TR:RXR  DNA  mRNA
HPT-Axis QSAR Development
Comparison of Endpoints of T4 Synthesis Inhibition
NIS activity
TPO activity
Membrane protein transports iodine
into the follicular cell
TPO iodinates tyrosine and couples iodotyrosines to produce thyroid hormone
• Limited data on chemical
inhibitors of NIS - mostly
monovalent anions of similar size
as iodide
• TPO inhibition data available for more
chemicals & classes of chemicals
• Methimazole – PTU
• Flavonoids
• Resorcinols
• Lack of data makes it difficult to
make informed chemical selection
• Difficult assay to transform to high
throughput format
• More data aids chemical selection
process and QSAR model development
• Spectrophotometric determination of
iodination of tyrosine to MIT
• Potential for conversion to high
throughput assay
HPT-Axis QSAR Development
TPO Inhibitors
Methimazole
CH3
Plant Flavonoids
O
flavone
O
N
OH
S
OH
NH
myricetin
O
HO
OH
OH
HO
Propylthiouracil
O
Resorcinol & Derivatives
OH
N
recorcinol
HS
OH
N
CH3
HO
Thyroid Peroxidase Inhibition
Literature Data
IC50 (M)
10-4
10-5
PTU
PTU
10-6
MM1
-1
0
1
2
3
log Kow
4
5
6
HPT-Axis QSAR Development
 Develop Xenopus-based in vitro assay to begin to test
known inhibitors of TPO activity
 Expand the range of chemicals and classes
 Select from EPA Chemical Lists
 Predictive Linkages
in vitro
→
ex vivo (explant culture)
→ in vivo
Systems Approach to Predicting Thyroid Toxicity
Chemical
Molecular
Effects
Biological Responses
Tissue ------------ Organism
Gene Expression
QSAR
Enzyme Activities
TPO
UDPGT
Regulatory Pathways
T4 synthesis and release
Feedback mechanisms
Adverse Effect
&
Compensatory
Response
Protein Binding
TR
Transthyretin
Serum Albumin
Ranking & Prioritization
of Chemicals
EPA Chemical
Lists
Selection for
Screening
MED Thyroid Project Team
S. Degitz
J. Tietge
J. Nichols
G. Holcombe
P. Kosian
D. Hammermeister
J. Korte
S. Batterman
B. Butterworth
M. Hornung
K. Thoemke
J. Chowdhury
J. Serrano
H. Kerr
L. Korte
M. Bugge
J. Olson
J. Haselman